1. Field of the Invention
This invention relates to the modification of plant gene expression causing modification of cell wall metabolism in plants.
2. Description of the Related Art
The modification of plant gene expression has been achieved by several methods. The molecular biologist can choose from a range of known methods to decrease or increase gene expression or to alter the spatial or temporal expression of a particular gene. For example, the expression of either specific antisense RNA or partial (truncated) sense RNA has been utilised to reduce the expression of various target genes in plants (as reviewed by Bird and Ray, 1991, Biotechnology and Genetic Engineering Reviews 9:207-227). These techniques involve the incorporation into the genome of the plant of a synthetic gene designed to express either antisense or sense RNA. They have been successfully used to down-regulate the expression of a range of individual genes involved in the development and ripening of tomato fruit (Gray et al, 1992, Plant Molecular Biology, 19:69-87). Methods to increase the expression of a target gene have also been developed. For example, additional genes designed to express RNA containing the complete coding region of the target gene may be incorporated into the genome of the plant to "over-express" the gene product. Various other methods to modify gene expression are known; for example, the use of alternative regulatory sequences.
The primary cell walls of fruit are important constituents that influence the physical and eating properties of fresh fruit and processed products. During fruit development and ripening, many biochemical changes occur that determine the composition, texture and quality of the ripe fruit. Fruit cell walls are continually modified by both synthetic and degradative processes as the fruit expand and eventually ripen. There are three major classes of polysaccharide in the cell wall: pectins, hemicelluloses and celluloses. These polysaccharide fractions undergo significant modifications in structure during ripening. Some of the most apparent of these occur in the pectic fraction including polyurinide solubilization, depolymerisation and a loss of wall arabinosyl and galactosyl residues (Gross and Wallner, 1979, Plant Physiol, 63:117-120; Gross, 1984, Physiol Plant, 62:25-32; Seymour et al, 1987, Phytochem, 26:1871-1875). Several pectin degrading enzymes have been isolated from tomato, including polygalacturonase and pectinesterase. Another important group of pectolytic enzymes are those which degrade the neutral sugar component, where galactosyl and arabinosyl residues occur as sidechains attached to a rhamnosyul unit in the main galacturonan backbone. Specific information on the structure of these sidechains is sparse due to their complexity, but in many plant tissues, including those from tomato fruit (Seymour et al, 1990, Phytochemistry, 29:725-731), chains of (1-&gt;4)-.beta.-D-galactan and (1-&gt;5) -linked arabinofuranosyl units are common (Carpita and Gibeaut, 1993, Plant J, 3:1-30). Fleshy fruits, including tomato, show a loss of wall galactosyl residues during ripening (Bartley, 1976, Phytochem, 15:625-626; Gross,1984, Physiol Plant, 62:25-32; Gross and Sams, 1984, Phytochemistry, 23:2457-2461; Redgwell et al, 1992, Plant Physiol, 98:71-81) and in tomato and kiwifruit, these changes are accompanied by an accumulation of free galactose (Gross, 1983, Phytochem, 22:1137-1139; Ogawa et al, 1990, J Jap Soc Food Sci, 37:298-305). The increased loss of cell wall galactosyl residues during ripening appears to be the result of increased solubilisation of galactosyl residues from the cell wall (Kim et al, 1991, Postharvest Biology and Technology, 1:67-80; Seymour et al, 1990, Phytochemistry, 29:725-731). The loss of cell wall galactose has also been reported in non-fruit tissues such as senescencing carnation petals (de Vetten and Huber, 1990, Physiol Plant, 78:447-454). Evidence indicates that these events are likely to result from the action of a .beta.-galactosidase/galactanase which acts on galactan-rich pectins.
.beta.-galactosidase activity has been detected in fruits including apple (Bartley, 1976, Phytochem, 15:625-626; Dick et al, 1990, Physiol Plant, 80:250-256), avocado (De Veau et al, 1993, Physiol Plant, 87:279-285), kiwifruit (Ogawa et al, 1990, J Jap Soc Food Sci Tech, 37:298-305; Ross et al, 1993, Planta, 189:499-506), muskmelon (Ranwala et al, 1992, Plant Physiology, 100:1318-1325) and tomato (Pressey, 1983, Plant Physiology, 71:132-135). In several fruit, including apple (Bartley IM, 1990, Phytochemistry, 13:2107-2111) and pepper (Gross et al, Physiol Plant, 66:31-36) there are large increases in galactosidase activity during ripening. However, in most cases the natural substrates for the galactosidase activity have not been identified.
In tomato three different .beta.-galactosidase activities were detected and partially purified, but only one of the isoforms (.beta.-galactosidase II, which increased in activity during ripening) could degrade a (1-&gt;4)-.beta.-D-galactan isolated from tomato cell walls (Pressey, 1983, Plant Physiology, 71:132-135; Pressey and Himmelsbach, 1984, Carbohydr Res, 127:356-359). The enzyme was therefore identified as an exo-(1-&gt;4)-.beta.-D-galactanase. Detailed studies on the composition and structure of tomato cell wall polysaccharhides by Seymour et al (1990, Phytochemistry, 29:725-731) demonstrated that galactans with this linkage predominate in tomato and undergo degradation during ripening. The free galactose measured in ripening tomato fruit is likely to result from this hydrolytic activity rather than altered metabolic utilization (Kim et al, 1991, Postharvest Biology and Technology, 1:67-80). The role of galactanase in tomato fruit ripening remains unknown, but it may play a key part in fruit softening. Galactan degrading enzymes have also been isolated from ripening avocado (De Veau et al, 1993, Physiol Plant, 87:279-285) and kiwifruit (Ross et al, 1993, Planta, 189:499-506).
Pectic polysaccharides in fruit consist mainly of an .alpha.-1,4-galactosyluronic acid backbone with 2- and 2,4-linked rhamnosyl residues interspersed in the chain (McNeil et al, 1984, Ann Rev Biochem, 53:625-663). The 2,4-linked rhamnosyl residues are thought to act as attachment points for sidechains of .beta.1,4-galactosyl and .alpha.1,5-linked arabinosyl residues (Seymour et al, 1990, Phytochemistry, 29:725-731). The role of the galactan rich side chains in the cell wall has not been confirmed. However, it is likey that they increase the interactions between cell wall components. Several mechanisms for these interactions are possible, for example: covalent links between galacturonan polymers; non-covalent interaction of pectin side chains; interactions with other cell wall components. The loss of galactosyl side chains from the pectin coincides with the major changes in texture that occur during fruit ripening.